Impact of Media in Transport Study on Cell Monolayer Integrity and Permeability

Literature review shows that various media have been used for in vitro transport study, but their impact on the monolayer integrity and permeability has been somehow neglected, and there is no systemic study on this subject. This study aims to investigate the impact of 6 commonly used media on Madin-Darby Canine Kidney cell viability and adherence, and the permeation of mannitol and propranolol. Dulbecco's modified Eagle medium (DMEM), cMedium (DMEM with serum), Hanks' balanced salt solution (HBSS), HBSS^- (without Ca²⁺/Mg²⁺), Dulbecco's phosphate-buffered saline (DPBS), and DPBS^- (without Ca²⁺/Mg²⁺) exhibited no cytotoxicity. Cell detachment was observed in 4 media in the order of DPBS^- ≈ HBSS^- > DPBS > HBSS. HBSS^-/DPBS^- caused 70% TEER reduction and 4-fold higher apparent permeability (P_app) of mannitol than the other media. The addition of D-glucose in DPBS^- decreased the P_app of mannitol. Cations and glucose negligibly influenced the P_app of propranolol. The addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in HBSS increased the P_app of both mannitol and propranolol for 2-3 folds without TEER reduction. In conclusion, the media compositions (Ca²⁺/Mg²⁺, glucose, vitamins, amino acids, and HEPES) can affect the monolayer integrity and permeability. DMEM and cMedium without HEPES are hence suggested as the media in transport study to avoid the potential impact on the monolayer integrity and permeability by the media.